An anti-platelet pharmaceutical composition and uses thereof

Abstract

The application discloses an anti-platelet drug composition, which comprises at least one of an FTase inhibitor and a GGTase inhibitor. The application also discloses application of the drug composition in preparation of an anti-platelet drug. The application proves that the MVA-GGPP pathway is a core axis for regulating platelet activation, and the FTase and GGTase inhibitors can inhibit synthesis of isoprene intermediates in the MVA-GGPP pathway, block protein isoprene modification mediated by the MVA-GGPP pathway, thereby inhibit the platelet activation level, and specifically eliminate the activation difference between MVK-deficient platelets and normal platelets. The FTase and GGTase inhibitors can be used as the anti-platelet drug, and can be used for prevention and treatment of thrombus-related diseases such as venous thrombosis and arterial thrombosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, particularly to the prevention and treatment of thrombotic diseases, and more specifically, to antiplatelet drugs. Background Technology

[0002] Platelet activation relies on the synergistic regulation of multiple signaling pathways and key proteins. Among these, membrane surface receptors, redox proteins, and inflammatory signaling pathways are the core targets mediating platelet adhesion, aggregation, and thrombus formation. Existing research has confirmed that PDI protein, ROS / NF-κB signaling pathway, and αIIbβ3 integrin receptor play key regulatory roles in the platelet activation cascade: under pathological stimulation, abnormal activation of these targets can significantly promote changes in platelet morphology, particle release, and expression of membrane surface adhesion molecules, accelerating platelet aggregation and thrombus formation, which is an important molecular mechanism for inducing arterial and venous thrombosis.

[0003] In the progression of diseases such as atherosclerosis, ischemic stroke, and deep vein thrombosis, vascular microenvironment disturbances continuously activate the aforementioned platelet activation pathways, leading to a hyperactivated platelet state and significantly increasing the risk of thrombosis and embolism. Currently, most traditional antiplatelet drugs in clinical practice inhibit downstream aggregation pathways, failing to precisely block the core signaling axis of platelet activation upstream. Their effectiveness in inhibiting persistent and refractory platelet activation is limited, and they easily disrupt the body's normal coagulation homeostasis, leading to a high incidence of bleeding complications.

[0004] Currently, intervention programs targeting single platelet targets generally suffer from limited regulatory dimensions and incomplete inhibitory effects. There is still no mature technology capable of precisely blocking core activation pathways, inhibiting pathological platelet activation, and maintaining physiological hemostasis. Therefore, developing novel targeted regulatory technologies, drugs, or formulations based on the mechanisms of action of key platelet activation targets and signaling pathways has significant clinical value and technological innovation for overcoming refractory thrombotic diseases and reducing treatment side effects.

[0005] The mevalonate (MVA) pathway is a core metabolic pathway in cells, which includes two major functional branches: (1) MVA-cholesterol branch: responsible for cell cholesterol synthesis and maintaining cell membrane lipid homeostasis; (2) MVA-GGPP (mevalonate-geranylgeranyl pyrophosphate) isoprene intermediate branch: synthesizes farnesyl pyrophosphate and geranylgeranyl pyrophosphate, mediates post-translational isoprene modification of small GTPase proteins, and regulates protein membrane localization, membrane anchoring and molecular function.

[0006] Current research generally believes that the MVA pathway regulates cell function mainly through the cholesterol synthesis branch, and assumes that cholesterol synthesis is the main pathway by which the MVA pathway affects platelet function. Statins lower cholesterol by inhibiting the rate-limiting enzyme in the upstream of the MVA pathway, and are used to lower lipids and indirectly and mildly inhibit platelet function. However, they do not distinguish the independent regulatory effects of the two pathway branches on platelet activation. Summary of the Invention

[0007] One of the technical problems to be solved by the present invention is to provide an antiplatelet drug composition that can effectively inhibit platelet overactivation mediated by the MVA-GGPP pathway.

[0008] To solve the above-mentioned technical problems, the antiplatelet drug composition of the present invention contains at least one of FTase (farnesyltransferase) inhibitor and GGTase (geranylgeranyltransferase) inhibitor.

[0009] The FTase inhibitor is preferably FTI277, and the GGTase inhibitor is preferably GGTI298.

[0010] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipients.

[0011] The dosage form of the pharmaceutical composition may be an intravenous injection, an intravenous infusion preparation, a tablet, or a capsule.

[0012] The second technical problem to be solved by the present invention is to provide the application of the above-mentioned pharmaceutical composition in the preparation of antiplatelet drugs.

[0013] The antiplatelet drug is used to prevent or treat thrombotic diseases caused by excessive platelet activation mediated by the MVA-GGPP pathway.

[0014] This invention demonstrates that the mevalonate-geranyl-geranyl pyrophosphate (MVA-GGPP) pathway is a core axis regulating platelet activation. FTase inhibitors and GGTase inhibitors can inhibit the synthesis of isoprene intermediates in the MVA-GGPP pathway and block MVA-GGPP pathway-mediated protein isoprene modification, thereby significantly inhibiting agonist-induced platelet aggregation, α-granule secretion, αIIbβ3 integrin activation, and P-selectin membrane exposure, thus suppressing platelet activation levels. These inhibitors can serve as specific antiplatelet drugs for the precise prevention and treatment of thrombosis-related diseases such as venous thrombosis and arterial thrombosis. Furthermore, the antiplatelet drugs of this invention specifically target FTase and / or GGTase, therefore only blocking abnormal platelet activation without affecting systemic cholesterol metabolism, resulting in a lower bleeding risk compared to statins. Attached Figure Description

[0015] Figure 1 This is a comparison of the aggregation levels of normal platelets and platelets treated with 5 μM GGTI298 under 0.05 U / mL thrombin induction, as shown in Example 1.

[0016] Figure 2 This is a comparison of the aggregation levels of normal platelets and platelets treated with 5 μM FTI277 under 0.05 U / mL thrombin induction, as shown in Example 2.

[0017] Figure 3 This is a comparison of the activation levels of normal platelets and platelets treated with 5 μM GGTI298 in Example 3. In Figure A, the P-selectin exposure level is shown, and in Figure B, the PAC1 antibody binding level is shown.

[0018] Figure 4 This is a comparison of the activation levels of normal platelets and platelets treated with 5 μM FTI277 in Example 4. In Figure A, the P-selectin exposure level is shown, and in Figure B, the PAC1 antibody binding level is shown.

[0019] Figure 5 This is a comparison of the spreading levels of normal platelets and platelets treated with 5 μM GGTI298 in Example 5.

[0020] Figure 6 This is a comparison of the spreading levels of normal platelets and platelets treated with 5 μM FTI277 in Example 6. Detailed Implementation

[0021] To provide a more detailed understanding of the technical content, features, and effects of the present invention, the technical solution of the present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0022] Example 1: Platelet activity assay after treatment with GGTase inhibitor I. Experimental Grouping The experiment in this embodiment was divided into a DMSO control group and a GGTI298 group.

[0023] II. Main Experimental Materials GGTase-specific inhibitor: GGTI298, manufactured by Selleck, catalog number S7466.

[0024] Platelet agonist: 0.05 U / mL thrombin.

[0025] III. Platelet Aggregation Test Method (1) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (2) DMSO and 5 μM GGTI298 were added to the resting human washed platelets of the corresponding groups, and the activity of platelets in different groups was evaluated after incubation at 37°C for 30 minutes.

[0026] (3) The washed platelets from different groups were placed in the aggregation channel in sequence, and the platelets were activated by 0.05 U / mL thrombin stimulation. The aggregation curves were analyzed and statistically analyzed.

[0027] The results are as follows Figure 1 As shown, the platelet aggregation level of GGTI298-treated platelets was significantly lower than that of control platelets.

[0028] Example 2: Platelet activity assay after treatment with FTase inhibitor I. Experimental Grouping The experiment in this embodiment was divided into a DMSO control group and an FTI277 group.

[0029] II. Main Experimental Materials FTase-specific inhibitor: FTI277, manufactured by Selleck, catalog number S7465.

[0030] Platelet agonist: 0.05 U / mL thrombin.

[0031] III. Platelet Aggregation Test Method (1) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (2) DMSO and 5 μM FTI277 were added to the resting human washed platelets of the corresponding groups, and the activity of platelets in different groups was evaluated after incubation at 37°C for 30 minutes.

[0032] (3) The washed platelets from different groups were placed in the aggregation channel in sequence, and the platelets were activated by 0.05 U / mL thrombin stimulation. The aggregation curves were analyzed and statistically analyzed.

[0033] The results are as follows Figure 2 As shown, the platelet aggregation level of FTI277-treated platelets was significantly lower than that of control platelets.

[0034] Example 3: GGTase Inhibitor Intervention Experiment I. Experimental Grouping The experiment in this embodiment was divided into a DMSO control group and a GGTI298 group.

[0035] II. Main Experimental Materials GGTase-specific inhibitor: GGTI298, manufactured by Selleck, catalog number S7466.

[0036] Platelet agonist: 0.05 U / mL thrombin.

[0037] P-selectin antibody: manufactured by BD Biosciences, catalog number 555524.

[0038] PAC1 antibody: manufactured by Thermo Fisher Scientific, catalog number MA5-28564.

[0039] III. Experimental Methods (1) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (2) DMSO and 5 μM GGTI298 were added to the resting human washed platelets of the corresponding groups, and the platelet activity of the DMSO group and the GGTI298 group was evaluated after incubation at 37°C for 30 minutes.

[0040] (3) Dilute the platelet concentrations of different groups to 1×10⁻⁶. 7 For each platelet sample, 100 μL of platelets was collected and divided into resting and activated groups. 0.05 U / mL thrombin was added to the activated platelet sample. Then, 1 μL of P-selectin antibody or 2 μL of PAC1 antibody was added to each 100 μL platelet sample. After incubation at 37°C for 30 minutes, the samples were analyzed by flow cytometry.

[0041] The results are as follows Figure 3 As shown, compared with the control group, treatment of normal platelets with 5 μM GGTI298 significantly inhibited agonist-induced platelet activation.

[0042] Example 4: FTase inhibitor intervention experiment I. Experimental Grouping The experiment in this embodiment was divided into a DMSO control group and an FTI277 group.

[0043] II. Main Experimental Materials FTase-specific inhibitor: FTI277, manufactured by Selleck, catalog number S7465.

[0044] Platelet agonist: 0.05 U / mL thrombin.

[0045] P-selectin antibody: manufactured by BD Biosciences, catalog number 555524.

[0046] PAC1 antibody: manufactured by Thermo Fisher Scientific, catalog number MA5-28564.

[0047] III. Experimental Methods (1) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (2) DMSO and 5μM FTI277 were added to the resting human washed platelets of the corresponding groups, and the activity of platelets in the DMSO group and FTI277 group was evaluated after incubation at 37°C for 30 minutes.

[0048] (3) Dilute the platelet concentrations of different groups to 1×10⁻⁶. 7 For each platelet sample, 100 μL of platelets was collected and divided into resting and activated groups. 0.05 U / mL thrombin was added to the activated platelet sample. Then, 1 μL of P-selectin antibody or 2 μL of PAC1 antibody was added to each 100 μL platelet sample. After incubation at 37°C for 30 minutes, the samples were analyzed by flow cytometry.

[0049] The results are as follows Figure 4 As shown, compared with the control group, treatment of normal platelets with 5 μM FTI277 significantly inhibited agonist-induced platelet activation.

[0050] Example 5: Platelet spreading assay of human blood platelets treated with GGTase inhibitor I. Experimental Grouping The experiment in this embodiment was divided into a DMSO control group and a GGTI298 group.

[0051] II. Main Experimental Materials GGTase-specific inhibitor: GGTI298, manufactured by Selleck, catalog number S7466.

[0052] Fibrinogen (Fg): Manufactured by Sigma-Aldrich, catalog number F3879, with a recommended concentration of 50 μg / mL.

[0053] TRITC Phalloidin (Rhodamine-labeled phalloidin): Manufactured by Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number 40734ES75.

[0054] III. Experimental Methods (1) Place the circular climbing slide in a 24-well plate, prepare a solution of Fg (pH 8.3 diluted with 0.1M NaHCO3) with a final concentration of 50 μg / mL in advance, add 300 μL of Fg solution on top of the climbing slide, and place it in a 4℃ refrigerator overnight. (2) Wash the smears with 1×PBS (for cells) three times, for 5 minutes each time; (3) Add 300 μL of 2% BSA to each well and seal at room temperature for 1 hour; (4) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (5) DMSO and 5 μM GGTI298 were added to the resting human washed platelets of the corresponding groups, and the platelet activity of the different groups was evaluated after incubation at 37°C for 30 minutes. (6) Adjust the concentration of the washed platelets after incubation to 1×10⁻⁶. 7 Add 300 μL of platelets per well and incubate at 37°C for 1 to 2 hours. Observe the morphology of the platelets under a microscope. If most of them appear as spread-out round or egg-shaped discs, they are spread-out platelets, and the 24-well plate can be removed from the 37°C incubator.

[0055] (7) After incubation, remove the 24-well plate from the incubator, remove the platelets, slowly add Tyrode's buffer along the well wall, and after washing, add 500 μL of 4% paraformaldehyde to each well for overnight fixation.

[0056] (8) Wash the slides with 1×PBS (for cells) three times for 5 minutes each time. Add 300 μL of the prepared labeling buffer to each well. After rupturing the membrane for 30 minutes, wash the slides with 1×PBS three times for 5 minutes each time.

[0057] (9) Add 300 μL of diluted Rhodamine-conjugated phalloidin antibody (diluted 1:800 with Labeling buffer) to each well, cover the 24-well plate with aluminum foil, and incubate at room temperature for 1 hour.

[0058] (10) Clean the slides with Tyrode's buffer three times, five minutes each time. After the last cleaning, remove the supernatant and remove the slides from the 24-well plate. Add an appropriate amount of anti-fluorescence quenching agent to the slide to fix the slides. Then observe the slides with a fluorescence microscope.

[0059] The results are as follows Figure 5As shown, treatment of normal platelets with GGTI298 significantly inhibited platelet spreading. This demonstrates that GGTase inhibitors can significantly suppress agonist-induced platelet activation.

[0060] Example 6: Spreading experiment of human platelets treated with FTase inhibitor I. Experimental Grouping The experiment in this embodiment was divided into a DMSO control group and an FTI277 group.

[0061] II. Main Experimental Materials FTase-specific inhibitor: FTI277, manufactured by Selleck, catalog number S7465.

[0062] Other materials are the same as in Example 5.

[0063] III. Experimental Methods The experimental method is the same as in Example 5, except that in step (5), 5μM FTI277 is used instead of 5μM GGTI298, and the other experimental steps are the same as in Example 5.

[0064] The results are as follows Figure 6 As shown, treatment of normal platelets with FTI277 significantly inhibited platelet spreading. This demonstrates that FTase inhibitors can significantly suppress agonist-induced platelet activation.

[0065] Example 7 Preparation of antiplatelet drug composition Take 5 mg of the active ingredient FTI277 or 5 mg of GGTI298 and 100 mg of mannitol, and prepare 1 mL of sterile intravenous injection solution with water for injection. After filtration and sterilization, it can be used for intravenous administration in various thrombosis model animals. It can significantly inhibit platelet activation in animals and reduce microvascular thrombosis.

[0066] The above embodiments are merely feasible or preferred embodiments of the present invention and are used to illustrate the present invention. They are not intended to limit the scope of the patent application of the present invention. Therefore, all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention should fall within the scope of the patent application of the present invention.

Claims

1. An antiplatelet drug composition, characterized in that, It contains at least one of FTase inhibitors and GGTase inhibitors.

2. The pharmaceutical composition according to claim 1, characterized in that, The FTase inhibitor is FTI277, and the GGTase inhibitor is GGTI298.

3. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

4. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The dosage form of the pharmaceutical composition is an intravenous injection, an intravenous infusion preparation, a tablet, or a capsule.

5. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of an antiplatelet drug.

6. The application according to claim 5, characterized in that, The antiplatelet drug is used to prevent or treat thrombotic diseases caused by excessive platelet activation mediated by the MVA-GGPP pathway.

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